Conformational Transitions at an S‐Layer Growing Boundary Resolved by Cryo‐TEM

S-layers are two-dimensional protein or glycoprotein lattices that cover the surfaces of many bacteria and archaea. Because they constitute the first interface of interaction between microorganisms and their environment, hosts, and predators, they are of great biological interest. Moreover, owing to their nanoscale, periodic, porous structure and relative ease of manipulation, they have the potential to be useful for both nano-biotechnological and materials applications. However, details of the assembly process are not yet known for any Slayer and high resolution structural information is very limited. Herein, we report a two-dimensional (2D) structural analysis of the expanding boundary of an isolated Lysinibacillus sphaericus S-layer (SbpA) growing on a graphene support. The results reveal previously unknown steps in the conformational transformation that drives the well-documented non-classical pathway of S-layer assembly and show how the fully-folded oligomeric repeating unit is entropically locked into the ordered array. In addition, our results provide the first demonstration that the unique physical properties of graphene offer superior image quality for cryogenic transmission electron microscopy (cryo-TEM) of biological macromolecules. S-layers assemble from a single protein or glycoprotein sequence to form a 2D lattice that covers the entire cell surface of microorganisms, including the cell poles and division sites. They are non-covalently attached to peptidoglycans and related polymers of gram-positive cell walls, linked to the outer membrane of gram-negative bacterial cell walls, and integrated into the cytoplasmic membrane through trans-membrane domains in gram-negative archaea. The primary sequence of the single protein or glycoprotein species contains all the information needed for assembly. Although they are often called “crystalline” cell surface layers, they are better described as “quasi-crystalline” or “paracrystalline”. S-layers also self-assemble in vitro in the presence of Ca ions, either on support films or in bulk solution, into ordered arrays with long-range order, substantially larger than a single cell. Previous studies found that self-assembly of SbpA (1268 residues from Lysinibacillus sphaericus) on lipid bilayers follows a multi-step pathway. It starts with the aggregation of monomers that adsorb onto the lipids in an extended conformation to form amorphous or liquid-like clusters. These clusters subsequently crystallize into the characteristic lattice of homotetrameric units, which grows by addition of new tetramers to the lattice edge sites. The rate of tetramer addition increases linearly with protein concentration, implying that monomers are added one at a time. However, the pathway through which the individual monomeric units become integrated with the correct conformation into the homotetrameric units—arguably the single most important step in S-layer assembly—remains unknown. To gain insight into S-layer assembly at the level of the tetramer subunits in the intact solution state, we obtained cryo-TEM images of single sheets, plunge-frozen while growing on graphene. Because the active self-assembling Slayers are instantly frozen, all the conformational states present at the expanding boundary on the graphene flat support are captured. Image alignment and averaging provide a view of the steps leading to subunit recruitment and maturation in S-layer self-assembly. For this study, we chose the surface layer protein SbpA (1268 residues), from the gram-positive bacterium Lysinibacillus sphaericus, which naturally forms a 2D quasi-crystalline cell envelope. We carried out the reconstitution of SbpA on single graphene sheets supported by TEM grids designed for cryo-TEM. The great potential of graphene for use as a support for biological cryo-TEM samples has recently been discussed. While mechanically strong and elastic, the 0.246 nm lattice constant and one-atom thickness of a single graphene layer, approximately 0.34 nm, make them trans[*] L. R. Comolli Life Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA 94720 (USA) E-mail: lrcomolli@lbl.gov